The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle

Summary Classification of human skeletal muscle into type I and type II fibres is frequently based on their weak or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in human muscle specimens with disease of the neuromuscular system.Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and gluconeogenesis respectively. Moreover the activities of enzymes of the pentose phosphate pathway are determined.A most important feature of the biochemical findings is that the constancy of activity ratios of the examined enzymes, as is found apparently normal human skeletal muscle, was frequently not present in diseased human skeletal muscle. From these results and from the histochemical results it can be concluded that for fibre classification in diseased human skeletal muscle the histochemical demonstration of myosin ATPase activity exclusively is not sufficient, but that it is necessary to apply other enzyme histochemical techniques too.Moreover it was found that in diseased human skeletal muscles the activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was strongly increased. A third observation was the relative decrease of the activity of the examined aerobic enzymes in affected muscle fibres of neurogenic muscle diseases.     

The foetal development of lactate dehydrogenase isoenzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from human striated muscle

1. Human foetal skeletal muscles involved in support and in periodic contractility were studied for their content of total extractable lactate dehydrogenase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities as well as for the relative distribution of lactate dehydrogenase isoenzymes. 2. During foetal development a linear steady increase in total lactate dehydrogenase activity as well as a linear decrease in the H/M sub-unit ratio of the isoenzymes was found. 3. No significant changes were found in the activities of the enzymes of the hexose monophosphate shunt (C-6 oxidation). 4. The changes found suggest a steady increased synthesis of lactate dehydrogenase M-sub-units in human skeletal muscles during foetal development. 5. The weekly changes in the total lactate dehydrogenase activity and in lactate dehydrogenase isoenzymes are lower in muscles involved in support than in those involved in periodic contractility. 6. These findings, together with the literature available, are consistent with the morphological fact that foetal development of skeletal muscles mostly concerns the white muscle fibres and not the red muscle fibres.     

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.     

Metabolic alterations in skeletal muscle of chronically streptozotocin-diabetic rats

This study was accomplished to determine the effects of chronic streptozotocin diabetes and insulin treatment on selected enzymes and substrates used in energy transduction in muscles composed of different muscle fiber types. Triglyceride concentration in all the muscles of diabetic rats was significantly elevated. Glycogen and protein concentrations were unchanged. The enzyme activities of hexokinase and alanine aminotransferase were significantly reduced and 3-hydroxyacyl-CoA dehydrogenase increased in all the muscles. Declines in phosphofructokinase, lactate dehydrogenase, citrate synthase, and succinate dehydrogenase activities were found in the red gastrocnemius and plantaris. Glycerol-3-phosphate dehydrogenase activity was lower than normal in the red gastrocnemius. Insulin treatment to the diabetic rats returned the altered triglyceride content and enzyme activities to normal, with exception of the lower alanine aminotransferase activity in the red gastrocnemius and plantaris. However, this enzyme was significantly ameliorated when compared with the untreated diabetic rats. The findings show that hypoinsulinism has a differential effect on the enzymatic profile of the different skeletal muscle fiber types, with those of the red gastrocnemius being most severely affected. Insulin treatment returned the enzymatic profile of the fiber types in diabetic rats to essentially normal.     

In vivo glycolytic equilibria in dog gracilis muscle

While the equilibrium assumption and the validity of using total measured concentrations for near equilibrium indicator reactions have been widely tested in liver, these have not been systematically evaluated in skeletal muscle. Vascularly isolated dog gracilis muscles were stimulated via the nerve at 4 Hz, and tissue was sampled by quick freezing at rest and after 10, 15, 30, 60, and 180 s of stimulation or after stimulation in the presence of glycolytic blockade by iodoacetate. Phosphocreatine, creatine, and several glycolytic intermediates were measured in tissue extracts. The in vivo mass action ratios for triosephosphate isomerase and aldolase were evaluated relative to substrate concentrations and compared with equilibrium constants determined in vitro. Although there was evidence of substrate binding at low substrate levels for the triosephosphate isomerase reaction, the in vivo mass action ratios for both reactions stabilized at a constant value at moderate substrate levels and in glycolytically blocked muscles. It was concluded that both enzymes are in apparent equilibrium in vivo, but the equilibrium constants are lower than those determined in vitro. The mass action ratios of the combined creatine kinase, lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase reactions were determined for resting muscles. These reactions are also at equilibrium and the equilibrium constants are consistent with in vitro values.     

Histochemical and electron microscopical studies of skeletal muscle affected by gas gangrene.

Ultrastructural and histochemical changes of skeletal muscle were studied in three patients affected with gas gangrene. There was complete lack of the phosphorylase, succinate dehydrogenase and adenosine triphosphatase activities in the affected muscles of all the patients. In unaffected muscles these enzymes showed weaker activities than in norm. The lactate dehydrogenase activity, especially the heart type isozyme (LDH-1 or H4) proved less sensitive to the effect of clostridial toxin. A general increase in the acid phosphatase activity was found both in affected and in unaffected muscles. On electron microscopic examination damage to sarcolemmal membrane and disintegration of myofilaments was seen. The mitochondria were swollen and their cristae distorted and fragmented.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue.

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used. Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine. The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 +/- 1% of leucine released from degradation of proteins in 30 subjects. A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed. The results indicate that biosynthesis and degradation of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly-turning-over proteins.     

Obesity and thyroid function. 3. Relationship between some indicators of thyroid function and the energy metabolism of striated muscle in obese women.

1. In a group of 23 obese women the relations between some indicators of thyroid function (thyroxine-binding globuline--T4BG, triiodothyronine-binding globuline--T3BG, Achilles tendon reflex--ART) on the one hand and activities of enzymes of the energy metabolism (hexokinase--HK, triose phosphate dehydrogenase--TPDH, lactate dehydrogenase--LDH, glycerol-3-phosphate dehydrogenase--GPDH, citrate synthease--CS, malate dehydrogenase--MDH, hydroxyacyl--COA dehydrogenase) in the quadriceps femoris muscle on the other hand were investigated. 2. Correlations were found between T4BG and TPDH, LDH and GPDH activities, between T3BG and TPDH and GPDH activities and between the value of the Achilles tendon reflex and TPDH activity. Functionally these enzymes activities are associated with glycolysis and hydrogen transport from cytoplasmatic NADH2. No correlations were found between enzymes of the aerobic metabolism incl. enzymes of fatty acid oxidation and indicators of thyroid function. 3. The results indicate a relationship between thyroid function and enzymes involved in glycolysis and hydrogen transport from cytoplasmatic NADH2. They do not suggest, however, the unequivocal conclusion that in obese women with reduced thyroid function there is a generally reduced energy supplying metabolism in skeletal muscle.     

Phosphomannosyl receptors of lysosomal enzymes in cardiac and skeletal muscles of young and old mice

The endogenous activity and the binding of high-uptake beta-N-acetylglucosaminidase were assayed in the membranes of heart and skeletal muscles of young (2 months) and old (15 months) NMRI-mice (Mus musculus) to evaluate the age-related changes in the phosphomannosyl receptors of lysosomal enzymes in muscular membranes. The total activities of beta-N-acetylglucosaminidase were significantly higher in cardiac and skeletal muscles of old than young mice. The total and the specific (inhibited by mannose-6-phosphate) binding of beta-N-acetylglucosaminidase to the membranes of cardiac muscle, but not to those of skeletal muscle, were higher in old mice than in young ones. The endogenous activity of beta-N-acetylglucosaminidase was significantly higher in the membranes of skeletal muscles of old mice than in those of young mice. The membranes of heart muscles did not show any difference in the endogenous activities. The saturation properties of the binding of beta-N-acetylglucosaminidase to the phosphomannosyl receptors were very similar in the membranes of heart and skeletal muscles of both age groups. We conclude that during aging the number of phosphomannosyl receptors of lysosomal enzymes increases in the membranes of heart muscle while the occupancy of phosphomannosyl receptors with endogenous ligands increases in the membranes of skeletal muscle.     

The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle

1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.     

Metabolic characteristics of the myocardium and muscle tissue of the thigh in rats

Myocardium and skeletal muscle of white rats have a number of specific features in metabolism of carbohydrates. The skeletal muscle is characterized by high intensity of glycolytic processes and glycolytic substrate phosphorylation, that is testified to by the activity of the terminal glycolysis stage enzymes (pyruvate kinase, lactate dehydrogenase, its isoenzyme spectrum) and by the content of lactate and pyruvate metabolites. In contrast to skeletal muscles, the activity of NAD-dependent malate dehydrogenase in the myocardium is significant both in cytoplasm and in mitochondria. This activity corresponds to a high level of malate and oxaloacetate metabolites and to the activity of NADP-dependent malate dehydrogenase, playing a connective role between glycolysis, the cycle of tricarboxylic acids and glyconeogenesis. Phosphoenolpyruvate carboxykinase, catalyzing the transformation of cytoplasmatic oxaloacetate into phosphoenolpyruvate is more active in the skeletal muscles where the intensity of the tricarboxylic acids cycle reactions is lower and the activity of glycolysis is higher than that of myocardium.     

Activity patterns of phosphofructokinase,glyceraldehydephosphate dehydrogenase,lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle

Summary Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

A comparative study of glycolysis in red and white muscles of the trout (Salmo gairdneri) and mirror carp (Cyprinus carpio)

The activities of some glycolytic and associated enzymes have been determined in the muscles of trout and carp to investigate the possibility that the discrepancies previously reported between lactate accumulation and anoxic tolerance in these two fish result from underlying differences in glycolytic potential. Steady state concentrations of certain glycolytic intermediates were also determined in freeze-clamped muscles from tankrested fish. The activities of hexokinase, phosphorylase and phosphofructokinase were approximately 2–3 times lower in carp than trout white muscles. Pyruvate kinase and lactate dehydrogenase activities were 5 times lower in carp white muscle. The lower, broader pH optima of lactate dehydrogenase and pyruvate kinase from carp compared to trout muscles is thought to be correlated with the greater anoxic tolerance of the carp. Glycolytic enzyme profiles were markedly different between the red and white muscles of the rainbow trout but broadly similar, with the exception of hexokinase activity, for the corresponding muscles of the carp. The results are discussed in relation to what is known about anaerobiosis in these two species and the comparative physiology of red and white muscles in fish.     

Skeletal muscle bioenergetics: a comparative study of mitochondria isolated from pigeon pectoralis, rat soleus, rat biceps brachii, pig biceps femoris and human quadriceps

The metabolism of mitochondria isolated from five functionally different skeletal muscles is compared. Data for a single ectothermic preparation are also reported. The mitochondria were prepared in yields of 44+/-7% from 50 to 100 mg muscle. The muscle content of mitochondrial protein ranged between 2 and 40 g kg(-1). Twelve specific activities of key enzymes and metabolic systems were determined, 10 of these in functional assays with respiratory measurements. The specific activities of glutamate dehydrogenase, alpha-glycerophosphate dehydrogenase, and exo-NADH oxidase differed considerably among muscle sources. Seven specific activities, including very central reactions, showed low among-muscle variation. The activity of ATP synthesis, for instance, was 1.0-1.3 mmol min(-1) g(-1) mitochondrial protein, 25 degrees C. In vitro data were extrapolated to in vivo conditions of the muscles. The calculated rates of respiration and ATP synthesis were in accordance with reported tissue activities. Pigeon pectoralis mitochondria showed a unique cytochrome spectrum and a respiratory chain activity that might effect simultaneous carbohydrate and fatty acid respiration. In mitochondria from the other muscles, the respiratory chain activity balanced the carbohydrate oxidation capacity. In all muscles, the respiratory capacity exceeds that needed for oxidative phosphorylation. This may secure maximal mitochondrial ATP synthesis during maximal work rates and high cellular [Ca(2+)].     

Effect of cadmium intoxication on glucose utilization in energy metabolism of muscles

The influence of cadmium intoxication on carbohydrate metabolism in skeletal muscles and liver of the male Wistar rats has been studied. Cadmium was administered as cadmium acetate in a dose of 0.3 mg Cd2+/kg body weight for three months. At the same time the control rats were injected with 0.9% NaCl. The animals were decapitated and samples of their skeletal muscles: the soleus muscle (composed mainly of red slow twitch fibers; ST) the gastrocnemius muscle containing two types of fibers (white fast twitch fibers FTb and red fast twitch fibers, FTa) and the liver were dissected out. In the samples of muscles, liver and serum contents of glycogen, glucose, pyruvate and lactate, as well as activities of hexokinase, pyruvate kinase and lactate dehydrogenase were measured. Intoxication of rats with cadmium for three months resulted in a reduction of glycolytic enzymes in the serum, ST and FTa muscle fibers and in the liver but did not change the activities of glycolytic enzymes in the FTb muscle fibers. The data obtained for the concentrations of glycogen in the liver and skeletal muscles suggest different mechanisms of cadmium influence on glycogen utilization in these organs.     

Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.     

Skeletal muscle lactate dehydrogenase isozyme alterations in men and women marathon runners

Total lactate dehydrogenase (LD) and LD isozyme activities in gastrocnemius muscle from trained men and women runners were measured in response to the chronic stress of training for a marathon race (42.2 km). Following 9 wk of training, total LD activity in skeletal muscle from men and women runners significantly (P less than 0.02) decreased 2.26 and 2.25 U/mg protein, respectively. However, men's total LD activities were significantly (P less than 0.001) less than the women's both before and after training. Significant (P less than 0.05) increases in LD1 activities in skeletal muscle in men and women runners were also observed after training. No significant correlations were detected between percent fiber type composition in men or women vs. the changes in total LD activity, changes in LD1 activity, maximal O2 consumption or training distance averaged per week after the training period. The biochemical adaptations in skeletal muscle that occurred in the LD isozyme composition in both men and women runners make the runners skeletal muscle appear similar to heart muscle in LD1 and LD2 activities.     

Physical training in man. Skeletal muscle metabolism in relation to muscle morphology and running ability.

The metabolic and morphologic adaptation to physical training in skeletal muscle tissue of eleven middle-aged, physically untrained men was studied. Muscle biopsies were taken from the vastus lateralis before, after 8 weeks and after 6 months of physical training for analysis of metabolic and morphologic variables. Glucose tolerance test indicated increased insulin sensitivity after 6 months of physical training. The activities of glycogen phosphorylase, hexokinase and glucose-6-P-dehydrogenase were increased but other enzymes involved in glycogen turnover and glycolysis were unchanged after 6 months of physical traning. The activities of citrate synthase and cytochrome-c-oxidase, representing the oxidative capacity were significantly increased already after 8 weeks of physical training. The incorporation rate of palmitate-carbon into CO2 and triglycerides increased, and the incorporation rate of leucine-carbon into CO2 decreased with 6 months of physical training. The fiber diameter of both Type 1- and Type 2-fibers increased, while the mitochondrial volume increased predominantly in Type 2-fibers. Significant correlations were found between metabolic, physiologic and morphologic variables before and after physical training. The results indicate an increased oxidative capacity, mainly located to Type 2-fibers, and an increased utilization of fatty acids in response to this type of physical training.